Cloning And Functional Analysis Of An Endsperm Specific Promoter Of ALP Type-B Gene

Cloning and functional analysis of wheat endosperm-specific promoters holds greatpromises. Along with providing a better understanding of the crop seed developmentprocesses and regulation mechanisms of crop endosperm-specific genes, it will help tostudy the gene modification including crop quality improvement and utilization of plantsas bioreactors in plant genetic engineering. Therefore, these promoters have potentialapplications both in theory and practical grounds.Avenin-like proteins (ALPs) genes are a new family of wheat storage protein genes, sonamed because of the similarity of their encoded protein sequences to Avenin proteins ofoats. ALP type-A and ALP type-B are two isoforms of avenin-like genes. Thedistinguishing feature of these proteins is that they include high levels of cysteine residues.ALP type-A proteins contain14cysteine residues. In contrast, ALP type-B proteinscontain at least18cysteine residues which are predicted to be able to form inter orintra-molecular disulfide bonds, thus raising the question of whether these cysteine-richproteins play a role in determining the functional properties of gluten.Furthermore, the expression of ALP type-B gene in endosperm were previouslyconfirmed by RT-PCR, but not in roots, stems or leaves, suggesting that the promoter hadendosperm-specificity. With the aims to get a wheat endosperm-specific promoter and tostudy its regulatory mechanism in the expression of storage protein, the upstream regionof ALP type-B gene was cloned and the expression of reporter gus gene-driven by it wasanalyzed in transgenic tobacco. The main results are as follows:(1) We successfully cloned a1,664bp upsteam sequence of ALP type-B from wheatEn1using Inverse-PCR (IPCR), based on the sequence of ALP type-B gene. Putativefunctional promoter elements were analyzed in PLACE and PlantCARE databases. Theresults showed that this upstream sequence contained the basic elements such asCAAT-box and TATA-box. Prolamin-box, GCN4-like motif (GLM), ESP-like element, RYmotif and G-box were also detected as endosperm-specific elements, indicating that thesemotifs may confer endosperm-specific expression for the promoter driven genes. It isworth mentioning that the ESP-like element at the position-677bp (ATG as+1) overlapswith an RY motif by four base pairs CATG, which is considered as a core sequence of the RY motif.(2) In order to analyze the function of various cis-elements in the expressionspecificity of ALP type-B gene promoter, as revealed by the results of the putativecis-element bioinformatics analysis, one vector named pALP17containing the1644bppromoter sequence and four other vectors named pALP-10, pALP-8, pALP-6, andpALP-3according to the length of the promoter respectively, with different5’-deletions:-999bp (ATG as+1),-785bp,-550bp, and-290bp were inserted into pBI121. ThepALP-3vector contained a prolamin-box which consists of an endosperm motif and GLMthat could drive a basal level of reporter gene and maintained the endosperm specificity.The pALP-6contained additional putative endosperm motifs and GLMs upstream of the–290bp prolamin-box, to check if they had any positive effect on endosperm-specificityof the promoter. The pALP-8contained the ESP-like element, which is the first report ofthis element in the promoter of any other species’ storage protein, since its discovery in oatglobulin promoter. The pALP-10contained G-box which is considered to havelong-distance positive effects. Agrobacteria LBA4404competent cells were transformedwith vectors pBI121, pALP-17, pALP-10, pALP-8, pALP-6, and pALP-3by thefreeze-thaw method. Then these vectors were introduced into the model plant tobacco(Nicotiana tobaccum L.) plants by Agrobacterium-mediated transformation method. Fivetransgenic tobacco plants for each of pALP-3, pALP-8and pALP-10respectively, fourfor pALP-6and nineteen for pALP-17were identified to be positive.(3) Histochemical GUS staining on T0transgenic tobacco plants showed that ALPtype-B promoter is indeed an endosperm-specific promoter which drove the expression ofreporter gene (gus) in endosperm but not in any other plant organs. Fluorescencequantitative analysis showed that although the-290bp promoter region had a weak basalGUS activity of273.06nmol/mg protein/hr, it was still nearly20fold high as comparedwith the negative control (14.25nmol/mg protein/hr) and could initiate theendosperm-specific expression. The-785bp promoter had the highest activity of447.86nmol/mg protein/hr, suggesting that the ESP-like element and RY motif might be playingin concert to drive the the expression of the reporter gene in the endosperm. While anobviously low level of GUS activity (173.68nmol/mg protein/hr) was observed when the length of the promoter was extended from-785to-1,664bp, suggesting that there shouldbe some positive or negative cis-elements in between these regions, and the G-box didn’thave long-distance positive effect in this promoter.In conclusion, this thesis reports the cloning and characterisation of anendosperm-specific promoter through stable transgenic experiments. Study of5’-deletionsof the promoter also lays foundations for the analysis and identification of newendosperm-specific cis-elements.